Project Overview: Cancer therapeutics targeted against the Epidermal Growth Factor Receptor (EGFR) have demonstrated great potential in lung cancer; however, these agents are effective in only a subset of patients. Furthermore, tumors tliat are initially responsive frequently acquire resistance over time. Though it is straightforward to measure molecular (DNA, RNA, protein) and biophysical (mass, density, charge) characteristics of tumors in bulk, recent studies have shown wide cell-tocell variability and the importance of characterizing that variability in estimating patient outcome^^^. We hypothesize that molecular and biophysical characterizations of circulating cells can discriminate cells that are responsive to therapy from those that are resistant. When analyzing cells collected from the circulation, or from other bodily fluids (e.g., pleural effusions, ascites), typically only a small number of cells are available. To asses the cell-to-cell heterogeneity of this limited number of cells, ive propose to develop and to apply quantitative, comprehensive single-cell analysis devices for assessing the DNA genome (e.g., single nucleotide polymorphisms, fusions, deletions), RNA expression, protein abundance (cell surface, intracellular, and secretome abundance), and biophysical properties of single cells for the dual purposes of predicting a patient's likely response to EGFR-targeted therapies and for monitoring a patient's acquisition of resistance to EGFR-targeted therapies (Fig. N3.3.1). We propose two specific aims for the development, testing, and application of our comprehensive analysis platform (Table N3.3.1).